The long-term goal of this application is to elucidate the mechanism(s) that regulate stress-induced and constitutive expression of heat shock proteins (hsps), which are critical not only for the cell's ability to survive exposure to stress conditions but also for normal protein folding in non-stressed cells. The stress-inducible vs. constitutive activities of the transcriptional regulatory proteins HSF1 and HSF2 are critical for this stress-activated and basal hsp gene expression, but how their differentially-regulated activities are controlled is unknown. In the search for this mechanism, we have identified differential regulation of SUMO-1 modification of HSF 1 and HSF2 leading to stress-induced and constitutive activation, respectively. We propose that SUMO-1 modification of HSFs is pre-requisite for their transcriptional activities at the levels of their ability to interact with promoters of hsp genes (DNA-binding activities), for assembly of transcription complexes with other factors on these promoters (transactivation potential), and for their protection against degradation. In this application we will 1) determine the functional consequences of SUMO-1 modification for the DNA-binding and transactivation activities of HSF 1 and HSF2, 2) characterize the role of this modification in regulating the turnover of HSF1 and HSF2, and 3) identify the mechanism(s) which mediate the differential regulation of stress-induced vs. constitutive SUMO-1 modification of HSF1 and HSF2, determine the significance of HSF sumoylation for protein misfolding in vivo, and determine whether sumoylation of HSFs is altered by cellular aging. Results from the proposed studies will define the basis of the differential regulation of HSF 1 and HSF2 leading to stress-induced and constitutive expression of heat shock proteins, and may provide a strategy for manipulating cellular heat shock protein expression, a promising potential treatment of diseases caused by protein misfolding/aggregation such as Parkinson's, Huntington's, and Alzheimer's Disease.